RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae.

Navas, Tony; Sánchez, Yolanda; Elledge, Stephen J.

doi:10.1101/gad.10.20.2632

Cited by 156 publications

(135 citation statements)

References 49 publications

(62 reference statements)

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“…The same study found that the mutator phenotype of the pol2-4 allele was unaffected by the absence of Dun1. This result is surprising, given the role of Pol e in helping to mediate the S-phase checkpoint (40)(41)(42)(43)(44). However, the weak pol2-4 mutator phenotype or the acquisition of a suppressor mutation may have limited the ability to detect a decrease in mutation rate in dun1Δ strains.…”

mentioning

confidence: 47%

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Williams

Marjavaara

Knowels

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) e mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol e depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol e proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol e base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol e mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.DNA replication and repair | polymerase fidelity | cancer | lethal mutagenesis

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mentioning

confidence: 47%

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Williams

Marjavaara

Knowels

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…7C). This could be explained by the fact that both Chk1 and Rad53 contribute to the DNA damage checkpoint downstream of Rad9, and Rad53 also requires Rad9 for signal amplification (20,21). Additionally, activated Chk1 may not be able to compensate for the loss of other Rad9 functions such as blocking processing of telomeres in cdc13-1 cells (22).…”

Section: Mec1mentioning

confidence: 95%

ATRMec1 Phosphorylation-independent Activation of Chk1 in Vivo

Chen

Caldwell

Pereira

et al. 2009

Journal of Biological Chemistry

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The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks. The current model is that after DNA damage or replication blocks, ATR Mec1 phosphorylates Chk1 on the non-catalytic C-terminal domain. However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus in vivo remains largely unknown. In this study we used an in vivo assay to examine the role of the C terminus of Chk1 in the response to DNA damage and replication blocks. The conserved ATR Mec1 phosphorylation sites were essential for the checkpoint response to DNA damage and replication blocks in vivo; that is, that mutation of the sites caused lethality when DNA replication was stalled by hydroxyurea. Despite this, loss of the ATR Mec1 phosphorylation sites did not change the kinase activity of Chk1 in vitro. Furthermore, a single amino acid substitution at an invariant leucine in a conserved domain of the non-catalytic C terminus restored viability to cells expressing the ATR Mec1 phosphorylation site-mutated protein and relieved the requirement of an upstream mediator for Chk1 activation. Our findings show that a single amino acid substitution in the C terminus, which could lead to an allosteric change in Chk1, allows it to bypass the requirement of the conserved ATR Mec1 phosphorylation sites for checkpoint function.Cell cycle checkpoints coordinate the maintenance of genomic integrity with cell division. The checkpoints that monitor replication fork integrity and DNA damage lesions sensed outside of DNA replication use similar mechanisms, and sometimes some of the same proteins, to delay cell cycle transitions, and in addition play an important role in the stability of replication forks and in DNA repair (1, 2).The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks in metazoans.

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“…Indeed, a link between pol and checkpoint activation, including the physical interaction between DinB and Hus1 and Rad1, was recently reported in Schizosaccharomyces pombe (49). Similarly, pol⑀ was reported to be a checkpoint protein (50). The experiments presented in this study provide evidence for the direct involvement of pol in TLS across BP-G adducts in living cells, since cells lacking pol exhibited a 3-fold reduction in TLS across this lesion, and expression of human pol in these cells resulted in complementation of the bypass defect.…”

Section: Bypass Of Bp-g Adducts By Pol In Mammalian Cellsmentioning

confidence: 99%

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

Avkin

Goldsmith

Velasco-Miguel

et al. 2004

Journal of Biological Chemistry

173

177

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Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol ). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol . Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol , suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.Genomic DNA is constantly subject to damage caused by both external agents, such as sunlight, and endogenous chemicals, such as reactive oxygen species. Most of this damage is eliminated by error-free DNA repair mechanisms, thereby restoring the DNA to its native sequence (1). However, a significant number of lesions escape repair and might therefore interfere with DNA replication and gene expression. Such interference can be mitigated by DNA damage tolerance mechanisms, primarily translesion DNA synthesis (TLS 1 ; also termed translesion replication) (2-5) and postreplicative recombinational repair (1, 6 -8). The key components in TLS are low fidelity DNA polymerases that specialize in lesion bypass (9 -12). These proteins were conserved in evolution and are present in organisms ranging from Escherichia coli to humans (13). Humans contain at least four specialized DNA polymerases belonging to the Y superfamily (pol , pol , pol , and REV1) as well as several from other polymerase families (e.g. pol (14), pol (15, 16), and pol (16, 17)). Many of these polymerases have been implicated in TLS in vitro (18 -24). However, there is a paucity of information about the efficiency and fidelity with which they support lesion bypass in living cells.Pol has a well established biological role in TLS, since it is mutated in all patients examined with the variant form of the hereditary disease xeroderma pigmentosum (10,18). This disease is characterized by sensitivi...

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RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae.

Cited by 156 publications

References 49 publications

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

ATRMec1 Phosphorylation-independent Activation of Chk1 in Vivo

Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

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